Fungal Biology and Biotechnology最新文献

{"title":"Class-II dihydroorotate dehydrogenases from three phylogenetically distant fungi support anaerobic pyrimidine biosynthesis.","authors":"Jonna Bouwknegt,&nbsp;Charlotte C Koster,&nbsp;Aurin M Vos,&nbsp;Raúl A Ortiz-Merino,&nbsp;Mats Wassink,&nbsp;Marijke A H Luttik,&nbsp;Marcel van den Broek,&nbsp;Peter L Hagedoorn,&nbsp;Jack T Pronk","doi":"10.1186/s40694-021-00117-4","DOIUrl":"https://doi.org/10.1186/s40694-021-00117-4","url":null,"abstract":"<p><strong>Background: </strong>In most fungi, quinone-dependent Class-II dihydroorotate dehydrogenases (DHODs) are essential for pyrimidine biosynthesis. Coupling of these Class-II DHODHs to mitochondrial respiration makes their in vivo activity dependent on oxygen availability. Saccharomyces cerevisiae and closely related yeast species harbor a cytosolic Class-I DHOD (Ura1) that uses fumarate as electron acceptor and thereby enables anaerobic pyrimidine synthesis. Here, we investigate DHODs from three fungi (the Neocallimastigomycete Anaeromyces robustus and the yeasts Schizosaccharomyces japonicus and Dekkera bruxellensis) that can grow anaerobically but, based on genome analysis, only harbor a Class-II DHOD.</p><p><strong>Results: </strong>Heterologous expression of putative Class-II DHOD-encoding genes from fungi capable of anaerobic, pyrimidine-prototrophic growth (Arura9, SjURA9, DbURA9) in an S. cerevisiae ura1Δ strain supported aerobic as well as anaerobic pyrimidine prototrophy. A strain expressing DbURA9 showed delayed anaerobic growth without pyrimidine supplementation. Adapted faster growing DbURA9-expressing strains showed mutations in FUM1, which encodes fumarase. GFP-tagged SjUra9 and DbUra9 were localized to S. cerevisiae mitochondria, while ArUra9, whose sequence lacked a mitochondrial targeting sequence, was localized to the yeast cytosol. Experiments with cell extracts showed that ArUra9 used free FAD and FMN as electron acceptors. Expression of SjURA9 in S. cerevisiae reproducibly led to loss of respiratory competence and mitochondrial DNA. A cysteine residue (C265 in SjUra9) in the active sites of all three anaerobically active Ura9 orthologs was shown to be essential for anaerobic activity of SjUra9 but not of ArUra9.</p><p><strong>Conclusions: </strong>Activity of fungal Class-II DHODs was long thought to be dependent on an active respiratory chain, which in most fungi requires the presence of oxygen. By heterologous expression experiments in S. cerevisiae, this study shows that phylogenetically distant fungi independently evolved Class-II dihydroorotate dehydrogenases that enable anaerobic pyrimidine biosynthesis. Further structure-function studies are required to understand the mechanistic basis for the anaerobic activity of Class-II DHODs and an observed loss of respiratory competence in S. cerevisiae strains expressing an anaerobically active DHOD from Sch. japonicus.</p>","PeriodicalId":52292,"journal":{"name":"Fungal Biology and Biotechnology","volume":"8 1","pages":"10"},"PeriodicalIF":0.0,"publicationDate":"2021-10-16","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC8520639/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"39523219","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"OA","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"Expanding the toolbox: another auxotrophic marker for targeted gene integrations in Trichoderma reesei.","authors":"Paul Primerano, Melani Juric, Robert Mach, Astrid Mach-Aigner, Christian Derntl","doi":"10.1186/s40694-021-00116-5","DOIUrl":"10.1186/s40694-021-00116-5","url":null,"abstract":"<p><strong>Background: </strong>The filamentous ascomycete Trichoderma reesei is used for the industrial production of cellulases and holds the promise for heterologous gene expression due to its outstandingly high protein secretion rates and its long-term application in industry and science. A prerequisite for successful heterologous gene expression is the ability to insert a corresponding expression cassette at suitable loci in the genome of T. reesei.</p><p><strong>Results: </strong>In this study, we test and demonstrate the applicability of the his1 gene [encoding for the ATP phosphoribosyltransferase (EC 2.4.2.17), part of the histidine biosynthesis pathway] and locus for targeted gene insertion. Deletion of the his1 promoter and a part of the coding region leads to histidine auxotrophy. Reestablishment of the his1 locus restores prototrophy. We designed a matching plasmid that allows integration of an expression cassette at the his1 locus. This is demonstrated by the usage of the reporter EYFP (enhanced yellow fluorescence protein). Further, we describe a minimal effort and seamless marker recycling method. Finally, we test the influence of the integration site on the gene expression by comparing three strains bearing the same EYFP expression construct at different loci.</p><p><strong>Conclusion: </strong>With the establishment of his1 as integration locus and auxotrophic marker, we could expand the toolbox for strain design in T. reesei. This facilitates future strain constructions with the aim of heterologous gene expression.</p>","PeriodicalId":52292,"journal":{"name":"Fungal Biology and Biotechnology","volume":"8 1","pages":"9"},"PeriodicalIF":0.0,"publicationDate":"2021-09-14","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC8442374/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"39415581","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"OA","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"Understanding and controlling filamentous growth of fungal cell factories: novel tools and opportunities for targeted morphology engineering.","authors":"Vera Meyer,&nbsp;Timothy Cairns,&nbsp;Lars Barthel,&nbsp;Rudibert King,&nbsp;Philipp Kunz,&nbsp;Stefan Schmideder,&nbsp;Henri Müller,&nbsp;Heiko Briesen,&nbsp;Anna Dinius,&nbsp;Rainer Krull","doi":"10.1186/s40694-021-00115-6","DOIUrl":"https://doi.org/10.1186/s40694-021-00115-6","url":null,"abstract":"<p><p>Filamentous fungal cell factories are efficient producers of platform chemicals, proteins, enzymes and natural products. Stirred-tank bioreactors up to a scale of several hundred m³ are commonly used for their cultivation. Fungal hyphae self-assemble into various cellular macromorphologies ranging from dispersed mycelia, loose clumps, to compact pellets. Development of these macromorphologies is so far unpredictable but strongly impacts productivities of fungal bioprocesses. Depending on the strain and the desired product, the morphological forms vary, but no strain- or product-related correlations currently exist to improve process understanding of fungal production systems. However, novel genomic, genetic, metabolic, imaging and modelling tools have recently been established that will provide fundamental new insights into filamentous fungal growth and how it is balanced with product formation. In this primer, these tools will be highlighted and their revolutionary impact on rational morphology engineering and bioprocess control will be discussed.</p>","PeriodicalId":52292,"journal":{"name":"Fungal Biology and Biotechnology","volume":"8 1","pages":"8"},"PeriodicalIF":0.0,"publicationDate":"2021-08-23","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC8383395/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"39337328","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"OA","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"Retraction Note to: Fungal sensing skin.","authors":"Andrew Adamatzky,&nbsp;Antoni Gandia,&nbsp;Alessandro Chiolerio","doi":"10.1186/s40694-021-00114-7","DOIUrl":"https://doi.org/10.1186/s40694-021-00114-7","url":null,"abstract":"","PeriodicalId":52292,"journal":{"name":"Fungal Biology and Biotechnology","volume":"8 1","pages":"7"},"PeriodicalIF":0.0,"publicationDate":"2021-06-02","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://sci-hub-pdf.com/10.1186/s40694-021-00114-7","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"39055379","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"OA","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"Towards fungal sensing skin.","authors":"Andrew Adamatzky, Antoni Gandia, Alessandro Chiolerio","doi":"10.1186/s40694-021-00113-8","DOIUrl":"10.1186/s40694-021-00113-8","url":null,"abstract":"<p><p>A fungal skin is a thin flexible sheet of a living homogeneous mycelium made by a filamentous fungus. The skin could be used in future living architectures of adaptive buildings and as a sensing living skin for soft self-growing/adaptive robots. In experimental laboratory studies we demonstrate that the fungal skin is capable for recognising mechanical and optical stimulation. The skin reacts differently to loading of a weight, removal of the weight, and switching illumination on and off. These are the first experimental evidences that fungal materials can be used not only as mechanical 'skeletons' in architecture and robotics but also as intelligent skins capable for recognition of external stimuli and sensorial fusion.</p>","PeriodicalId":52292,"journal":{"name":"Fungal Biology and Biotechnology","volume":"8 1","pages":"6"},"PeriodicalIF":0.0,"publicationDate":"2021-05-12","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC8117569/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"38905199","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"OA","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"An approach to change the basic polymer composition of the milled Fomes fomentarius fruiting bodies.","authors":"Liudmila Kalitukha","doi":"10.1186/s40694-021-00112-9","DOIUrl":"https://doi.org/10.1186/s40694-021-00112-9","url":null,"abstract":"<p><strong>Background: </strong>Chitin and its derivative chitosan are readily exploited, especially in food, cosmetic, pharmaceutical, biomedical, chemical, and textile industries. The biopolymers are currently recovered from the crustacean shells after purification from the large amount of proteins and minerals. The key problems are centered around a lot of chemical waste and allergenic potential of the heat-stable remaining proteins. Fungi can be considered as an alternative eco-friendlier source of the chitin and chitosan due to the lower level of inorganic materials and absence of the allergenic proteins.</p><p><strong>Results: </strong>The work presents a new chemical assay to change the composition of the milled Fomes fomentarius fruiting bodies. A gradual 13-fold increase of the chitin amount accompanied by 14-fold decrease of the glucan content was obtained after repetitive alkali-acidic treatment. Raw material contained mainly chitin with 30% degree of deacetylation. After the first and second alkali treatment, the polymer was defined as chitosan with comparable amounts of N-acetyl-D-glucosamine and D-glucosamine units. The last treated samples showed an increase of the chitin amount to 80%, along with typical for the natural tinder fibers degree of deacetylation and three-dimensional fibrous hollow structure.</p><p><strong>Conclusions: </strong>A new approach allowed a gradual enrichment of the pulverized Fomes fomentarius fruiting bodies with chitin or chitosan, depending on the extraction conditions. High stability and fibrous structure of the fungal cell walls with a drastically increased chitin ratio let us suggest a possibility of the targeted production of the chitin-enriched fungal material biotechnologically under eco-friendly conditions.</p>","PeriodicalId":52292,"journal":{"name":"Fungal Biology and Biotechnology","volume":"8 1","pages":"5"},"PeriodicalIF":0.0,"publicationDate":"2021-04-15","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://sci-hub-pdf.com/10.1186/s40694-021-00112-9","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"38877494","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"OA","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"Preservation stress resistance of melanin deficient conidia from Paecilomyces variotii and Penicillium roqueforti mutants generated via CRISPR/Cas9 genome editing.","authors":"Sjoerd J Seekles,&nbsp;Pepijn P P Teunisse,&nbsp;Maarten Punt,&nbsp;Tom van den Brule,&nbsp;Jan Dijksterhuis,&nbsp;Jos Houbraken,&nbsp;Han A B Wösten,&nbsp;Arthur F J Ram","doi":"10.1186/s40694-021-00111-w","DOIUrl":"https://doi.org/10.1186/s40694-021-00111-w","url":null,"abstract":"<p><strong>Background: </strong>The filamentous fungi Paecilomyces variotii and Penicillium roqueforti are prevalent food spoilers and are of interest as potential future cell factories. A functional CRISPR/Cas9 genome editing system would be beneficial for biotechnological advances as well as future (genetic) research in P. variotii and P. roqueforti.</p><p><strong>Results: </strong>Here we describe the successful implementation of an efficient AMA1-based CRISPR/Cas9 genome editing system developed for Aspergillus niger in P. variotii and P. roqueforti in order to create melanin deficient strains. Additionally, kusA^- mutant strains with a disrupted non-homologous end-joining repair mechanism were created to further optimize and facilitate efficient genome editing in these species. The effect of melanin on the resistance of conidia against the food preservation stressors heat and UV-C radiation was assessed by comparing wild-type and melanin deficient mutant conidia.</p><p><strong>Conclusions: </strong>Our findings show the successful use of CRISPR/Cas9 genome editing and its high efficiency in P. variotii and P. roqueforti in both wild-type strains as well as kusA^- mutant background strains. Additionally, we observed that melanin deficient conidia of three food spoiling fungi were not altered in their heat resistance. However, melanin deficient conidia had increased sensitivity towards UV-C radiation.</p>","PeriodicalId":52292,"journal":{"name":"Fungal Biology and Biotechnology","volume":"8 1","pages":"4"},"PeriodicalIF":0.0,"publicationDate":"2021-04-02","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://sci-hub-pdf.com/10.1186/s40694-021-00111-w","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"25539748","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"OA","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"Fungal sensing skin.","authors":"Andrew Adamatzky, Antoni Gandia, Alessandro Chiolerio","doi":"10.1186/s40694-021-00110-x","DOIUrl":"10.1186/s40694-021-00110-x","url":null,"abstract":"<p><strong>Background: </strong>A fungal skin is a thin flexible sheet of a living homogeneous mycelium made by a filamentous fungus. The skin could be used in future living architectures of adaptive buildings and as a sensing living skin for soft self-growing/adaptive robots.</p><p><strong>Results: </strong>In experimental laboratory studies we demonstrate that the fungal skin is capable for recognising mechanical and optical stimulation. The skin reacts differently to loading of a weight, removal of the weight, and switching illumination on and off.</p><p><strong>Conclusion: </strong>These are the first experimental evidences that fungal materials can be used not only as mechanical 'skeletons' in architecture and robotics but also as intelligent skins capable for recognition of external stimuli and sensorial fusion.</p>","PeriodicalId":52292,"journal":{"name":"Fungal Biology and Biotechnology","volume":"8 1","pages":"3"},"PeriodicalIF":0.0,"publicationDate":"2021-03-17","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC7972235/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"25488172","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"OA","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"A fully automated pipeline for the dynamic at-line morphology analysis of microscale Aspergillus cultivation.","authors":"Roman Jansen,&nbsp;Kira Küsters,&nbsp;Holger Morschett,&nbsp;Wolfgang Wiechert,&nbsp;Marco Oldiges","doi":"10.1186/s40694-021-00109-4","DOIUrl":"https://doi.org/10.1186/s40694-021-00109-4","url":null,"abstract":"<p><strong>Background: </strong>Morphology, being one of the key factors influencing productivity of filamentous fungi, is of great interest during bioprocess development. With increasing demand of high-throughput phenotyping technologies for fungi due to the emergence of novel time-efficient genetic engineering technologies, workflows for automated liquid handling combined with high-throughput morphology analysis have to be developed.</p><p><strong>Results: </strong>In this study, a protocol allowing for 48 parallel microbioreactor cultivations of Aspergillus carbonarius with non-invasive online signals of backscatter and dissolved oxygen was established. To handle the increased cultivation throughput, the utilized microbioreactor is integrated into a liquid handling platform. During cultivation of filamentous fungi, cell suspensions result in either viscous broths or form pellets with varying size throughout the process. Therefore, tailor-made liquid handling parameters such as aspiration/dispense height, velocity and mixing steps were optimized and validated. Development and utilization of a novel injection station enabled a workflow, where biomass samples are automatically transferred into a flow through chamber fixed under a light microscope. In combination with an automated image analysis concept, this enabled an automated morphology analysis pipeline. The workflow was tested in a first application study, where the projected biomass area was determined at two different cultivation temperatures and compared to the microbioreactor online signals.</p><p><strong>Conclusions: </strong>A novel and robust workflow starting from microbioreactor cultivation, automated sample harvest and processing via liquid handling robots up to automated morphology analysis was developed. This protocol enables the determination of projected biomass areas for filamentous fungi in an automated and high-throughput manner. This measurement of morphology can be applied to describe overall pellet size distribution and heterogeneity.</p>","PeriodicalId":52292,"journal":{"name":"Fungal Biology and Biotechnology","volume":"8 1","pages":"2"},"PeriodicalIF":0.0,"publicationDate":"2021-03-06","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://sci-hub-pdf.com/10.1186/s40694-021-00109-4","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"25441255","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"OA","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"Optimization of scleroglucan production by Sclerotium rolfsii by lowering pH during fermentation via oxalate metabolic pathway manipulation using CRISPR/Cas9.","authors":"Tianlong Bai,&nbsp;Teng Wang,&nbsp;Yan Li,&nbsp;Na L Gao,&nbsp;Lixin Zhang,&nbsp;Wei-Hua Chen,&nbsp;Xiushan Yin","doi":"10.1186/s40694-021-00108-5","DOIUrl":"https://doi.org/10.1186/s40694-021-00108-5","url":null,"abstract":"<p><strong>Background: </strong>Sclerotium rolfsii is a potent producer of many secondary metabolites, one of which like scleroglucan is an exopolysaccharide (EPS) appreciated as a multipurpose compound applicable in many industrial fields.</p><p><strong>Results: </strong>Aspartate transaminase (AAT1) catalyzes the interconversion of aspartate and α-ketoglutarate to glutamate and oxaloacetate. We selected AAT1 in the oxalate metabolic pathway as a target of CRISPR/Cas9. Disruption of AAT1 leads to the accumulation of oxalate, rather than its conversion to α-ketoglutarate (AKG). Therefore, AAT1-mutant serves to lower the pH (pH 3-4) so as to increase the production of the pH-sensitive metabolite scleroglucan to 21.03 g L^-1 with a productivity of up to 0.25 g L^-1·h^-1.</p><p><strong>Conclusions: </strong>We established a platform for gene editing that could rapidly generate and select mutants to provide a new beneficial strain of S. rolfsii as a scleroglucan hyper-producer, which is expected to reduce the cost of controlling the optimum pH condition in the fermentation industry.</p>","PeriodicalId":52292,"journal":{"name":"Fungal Biology and Biotechnology","volume":"8 1","pages":"1"},"PeriodicalIF":0.0,"publicationDate":"2021-02-18","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://sci-hub-pdf.com/10.1186/s40694-021-00108-5","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"25386120","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"OA","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}